Injectable solution of benzalkonium fluoride

ABSTRACT

PCT No. PCT/FR95/00070 Sec. 371 Date Oct. 22, 1996 Sec. 102(e) Date Oct. 22, 1996 PCT Filed Jan. 23, 1995 PCT Pub. No. WO95/19766 PCT Pub. Date Jul. 27, 1995Solution administrable by intravascular injection in a human or an animal which comprises an effective amount up to 0.23% by weight of a benzalkonium fluoride having the formula:   &lt;IMAGE&gt;   where R is an alkyl radical which may vary between C8H17 and C18H37, dissolved in an excipient injectable intravascularly. Preferably R is between C12H25 and C14H29, and more preferably C14H29. The solution can be packaged in 10 ml ampoules, containing about 21 mg of benzalkonium fluoride, the benzalkonium fluoride being dissolved in a physiological solvent. The solution also contains at least one metallic derivative of fluorine, preferably lithium fluoride.

This application is a 371 of PCT/FR95/00070, filed Jan. 23, 1995.

The invention concerns a solution which can be administeredintravascularly consisting of at least one benzalkonium fluoride.

EP-A-0,308,564 already mentions the use of benzalkonium fluoride toobtain a composition for general or parenteral administration withbetween 0.05% and 7%, more particularly of the order of 1% in weight ofbenzalkonium fluoride, intented to act against viruses and retroviruses,particularly herpes or the LVAs responsible for AIDS.

Nevertheless, benzalkonium fluoride is supposed, as with all thebenzalkonium salts which are tensio-active, to have a strong h.aebutted.molytic effect, prohibiting its intravascular administration.Thus, although general or parenteral administration of benzalkoniumfluoride are mentionned in EP-A-0,308,564, its use in practice comes upagainst this difficulty, until now considered insurmountable for tworeasons: the concentration to reach in the blood to arrive at anactivity was itself supposed to lead to h.ae butted.molysis of theblood; injection, even administered very slowly, supposes the localcreation of a high, thus h.ae butted.molytic, concentration ofbenzalkonium fluoride. In particular, injection of this salt atconcentrations of the order of 1% weight appeared to be impossible. Nowthis concentration was considered indispensable to obtain a sufficientactivity. We thus renounced the use of benzalkonium salts, andparticularly benzalkonium fluoride, by intravascular administration,considering that the concentrations of the injected solutions wouldsuppress all virucidal and bactericidal activity, taking into accountthe minimal inhibiting concentration measured in vitro, and that, evenat these low concentrations, a very high risk of h.ae butted.molysis,and thus immediate death, persists. Thus until now, intravascularadministration of benzalkonium salts, and notably benzalkonium fluoridein man or animals was considered purely and simply forbidden.

A constant need makes itself felt for a shock treatment against viral orinfectious affections, notably of immunodeficient origin, and inparticular in the treatment of patients suffering from AIDS in thedeclared or evolutive phase which present severe opportunisticinfections leading rapidly to dehydration and death.

Such a shock treatment poses a series of apparently contradictoryproblems, however:

it should act not only against the pathogenic micro-organismsresponsible for the deteriorated clinical state of the patient, butequally against the virus responsible for the immunodeficiency (the caseof AIDS),

it should have a very wide action spectrum, taking into account thegreat variety of affections which may be involved in the case of animmunodeficiency,

it should nevertheless be free of side effects, and in particular shouldnot irritate or attack healthy organs or cells in an alreadyconsiderably weakened organism.

The invention thus aims in a general way at finding a solution to thisgeneral problem which has not, until now, been resolved. It thus aims atoffering a treatment to patients suffering from viral or severeinfectious affections, particularly multiple ones, such as those due toan immunodeficiency.

More particularly, the inventors discovered with surprise that, amongthe quaternary ammoniums, benzalkonium fluoride can be effectivelyinjected intravascularly without risk of h.ae butted.molysis within aprecise range of concentrations with which an effective activity is,nevertheless, noted.

The invention thus aims more particularly at offering an injectablesolution of an antiviral or anti-infectious agent such as a benzalkoniumsalt.

For this, the invention concerns a solution administerableintravascularly, characterised by a content of the order of 0.23% orless in weight of a benzalkonium fluoride having the formula: ##STR2##where R is an alkyl radical which may vary between C₈ H₁₇ and C₁₈ H₃₇,dissolved in an excipient injectable intravascularly.

We could in effect note that when the proportions by weight ofbenzalkonium fluoride are of the order of 0.23% or less, and moreparticularly of the order of 0.20% of weight, no h.ae butted.molysis isto be feared in general, contrary to the previous general opinion of theart. Also, a clinical improvement can be noted in using concentrationsgreater than 0.05% of weight of benzalkonium fluoride. Thus the solutionaccording to the invention is between 0.05 and 2.3 g/l--moreparticularly of the order of 2.1 g/l--of benzalkonium fluoride,preferably a benzalkonium fluoride in which R is C₁₄ H₂₉.

More particularly, the solution according to the invention consists of aconcentration of benzalkonium fluoride determined to permitadministration by slow injection of unit doses of the order of 0.5 to 23mg--notably of the order of 21 mg--of benzalkonium fluoride per liter ofblood of the patient to be treated. Thus, in the case of a man havingfive liters of blood, we prepare an injected dose of 2.5 to 130 mg, moreparticularly of the order of 105 mg, of benzalkonium fluoride.

Thus, according to the invention, two conditions should be broughttogether to allow intravascular injection of benzalkonium fluoride: theconcentration of benzalkonium fluoride in the injected solution shouldbe less than 0.23% and the total quantity injected per liter of blood ofthe patient should remain inferior to 23 mg.

To determine the concentration of benzalkonium fluoride in the solutionto be injected, we take a blood sample from the patient immediatelybefore injection, submit this blood to in vitro h.ae butted.molytictests with a solution consisting initially of 0.23% of benzalkoniumfluoride, and reduce the concentrations until a non-h.ae butted.molyticsolution is obtained.

According to the invention, before injection we verify the non h.aebutted.molytic character of the solution by determining the h.aebutted.molytic concentration on the patient's blood.

The solution according to the invention may be packaged in ampoules from5 to 15 ml--more particularly 10 ml--consisting of between 0.5 and 23mg--more particularly 21 mg for a 10 ml ampoule--of benzalkoniumfluoride respecting the maximum value of 0.23%. And we inject between 5and 15 ml, more particularly 10 ml, of the solution per litre of blood,more particularly 50 ml in the case of a man.

The solution according to the invention advantageously consists ofbenzalkonium fluoride dissolved in a physiological solute. R fallsadvantageously between C₁₂ H₂₅ and C₁₄ H₂₉. According to the invention,the solution may contain, among others, at least a metallic fluorinederivative, and more particularly lithium fluoride.

The invention further relates to the application of at least abenzalkonium fluoride whose formula is given above, dissolved in aconcentration of the order of or less than 0.23% of weight in anexcipient injectable intravascularly for combatting viral or infectiousdisease, notably of an immunodeficient origin. More particularly, theinvention concerns at least such a benzalkonium fluoride dissolved at aconcentration of the order or less than 0.23% of weight in an excipientinjectable intravascularly to obtain a solution administerableintravascularly for combatting AIDS in the declared or evolutive phase.The invention thus relates to a method of treating viral or infectiousdiseases, notably of an immunodeficient origin, such as AIDS in thedeclared or evolutive phase in which we inject a solution according tothe invention intravascularly. In the method of the invention, we injecta solution after having determined ex vivo the h.ae butted.molyticpotential of benzalkonium fluoride vis-a-vis the patient's blood, thesolution injected having a concentration inferior to this h.aebutted.molytic concentration, and we administer by slow injection aquantity of benzalkonium fluoride between 0.5 and 23 mg per litre ofblood of the patient to be treated, more particularly and preferablybetween 20 and 23 mg.

Thus, in an application according to the invention, we use between 0.5and 23 mg--more particularly of the order of 21 mg--of benzalkoniumfluoride dissolved in 10 ml of solute.

The invention is therefore particularly useful in the treatment of viraldiseases which destroy the immune defences, and more particularly as ashock treatment. The invention has thus permitted effective treatment ofcases of Carre's disease and cases of AIDS in an advanced phase, that isdeveloping opportunistic infectious diseases.

Other characteristics and advantages of the invention will appear in theexamples given below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 given as appendices represent curves resulting from invitro measurement tests of virucide activity of on the HIV-1 virus ofbenzalkonium fluoride conforming to example 2 described below.

IN VITRO VIRUCIDE TRIALS Example 1

We have determined the virucide activity oftetradecyl-dimethyl-benzyl-ammonium fluoride (BKF) vis-a-vis vertebrateviruses in conformance with French Standard AFNOR T72-180, December1989. This benzalkonium fluoride was diluted in solution in steriledistilled water at the following concentrations: 0.02, 0.01, 0.005%(weight divided by volume).

The tests were carried out on the following viral strains:

picornavirid.ae butted.: polio enerovirus 1, SABIN strain, cultivated onVERO cell,

adenovirid.ae butted.: adenovirus type 5, cultivated on KB cell,

herpetovirid.ae butted.: Herpes Simplex Virus Type 1 (HSV 1) and HerpesSimplex Virus Type 2 (HSV 2) supplied by the Institut Pasteur andcultivated on cell MCR-5.

The water, the culture medium and the reagents are those recognised byStandard AFNOR T72-180. The titration of the virus is by reading thecytopathic test, following the method described in Standard AFNORT72-180. The calculation of the infectious titration is by the FISHERand YATES method with the WYSHAK and DETRE tables. The results are givenin number of infectious units per ml, or plaque-forming units per ml(PFU/ml).

We first sought the toxicity threshold (D.L.0) of benzalkonium fluoride.Before filtration, this toxicity threshold was 0.05% (in mg per ml).After filtration, the toxicity threshold was 0.01%.

The capacity of cells treated with benzalkonium fluoride to develop theviral infection was determined and the results a given in Table 1.

                  TABLE 1                                                         ______________________________________                                        CAPACITY OF CELLS TREATED BY BKF                                              TO DEVELOP THE VIRAL INFECTION                                                Titration of viral suspensions (PFU/ml)                                                                After contact                                                                 with benzaikonium                                    Strains          Control fluoride                                             ______________________________________                                        ENTEROVIRUS      10.sup.7.68                                                                           10.sup.6.98                                          POLIO 2                                                                       ADENOVIRUS       10.sup.8.64                                                                           10.sup.7.56                                          Type 5                                                                        HERPES SIMPLEX   10.sup.8.00                                                                           10.sup.7.02                                          VIRUS Type 2 (HSV 2)                                                          HERPES SIMPLEX   10.sup.8.50                                                                           10.sup.7.84                                          VIRUS Type 2 (HSV 2)                                                          ______________________________________                                    

The virucidal activity was then determined by the technique known asmolecular screening, consisting of filtration on sephadex gel LH 20 andusing subcytotoxic concentrations of benzalkonium fluoride. Thevirucidal activity was determined as a function of contact time betweenthe benzalkonium fluoride and the viral strain. Different samples weretaken at predetermined time intervals, submitted to a centrifugation for8 minutes at 1,000 g, then titrated on microplates. A control viralsuspension without benzalkonium fluoride was treated following the samemethodology. Tables 2 to 5 give the results obtained in kinetic terms ofthe inactivation of the virus as a function of contact time with thebenzalkonium fluoride and its concentration. The virucidal activity ofbenzalkonium fluoride (BKF) is determined by the minimal concentrationand minimal contact time with the virus which provoked a reduction ofinfectious titration of the viruses by a logarithmic factor of a least4. The tests were carried out at 32° C.

                  TABLE 2                                                         ______________________________________                                        VIRUCIDAL ACTIVITY OF BKF ON ENTEROVIRUS POLIO 1                              Viral Titration (PFU/ml)                                                      Concentration                                                                 of BKF       Virus/BKF contact time (minutes)                                 (%) (m/V)    15     30         60   120                                       ______________________________________                                        0.02         10.sup.3.28                                                                          10.sup.2.02                                                                              10.sup.1.40                                                                        0                                         0.01         10.sup.1.28                                                                          0          0    0                                         0.005        10.sup.4.38                                                                          10.sup.3.62                                                                              10.sup.3.02                                                                        10.sup.2.26                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        VIRUCIDAL ACTIVITY OF BKF ON ADENOVIRUS TYPE 5                                Viral Titration (PFU/ml)                                                      Concentration                                                                 of BKF       Virus/BKF contact time (minutes)                                 (%) (m/V)    15     30         60   120                                       ______________________________________                                        0.02         10.sup.5.88                                                                          10.sup.5.02                                                                              10.sup.4.16                                                                        10.sup.2.68                               0.01         10.sup.5.04                                                                          10.sup.4.38                                                                              10.sup.3.86                                                                        10.sup.3.02                               0.005        10.sup.6.00                                                                          10.sup.5.65                                                                              10.sup.5.10                                                                        10.sup.4.60                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        VIRUCIDAL ACTIVITY OF BKF ON HERPES SIMPLEX                                   VIRUS TYPE 1                                                                  Viral Titration (PFU/ml)                                                      Concentration                                                                 of BKF       Virus/BKF contact time (minutes)                                 (%) (m/V)    15     30         60   120                                       ______________________________________                                        0.02         0      0          0    0                                         0.01         10.sup.1.68                                                                          0          0    0                                         0.005        10.sup.4.26                                                                          10.sup.3.15                                                                              10.sup.2.27                                                                        10.sup.1.36                               ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        VIRUCIDAL ACTIVITY OF BKF ON HERPES SIMPLEX                                   VIRUS TYPE 2                                                                  Viral Titration (PFU/ml)                                                      Concentration                                                                 of BKF       Virus/BKF contact time (minutes)                                 (%) (m/V)    15     30         60   120                                       ______________________________________                                        0.02         0      0          0    0                                         0.01         10.sup.2.68                                                                          10.sup.1.41                                                                              0    0                                         0.005        10.sup.8.14                                                                          10.sup.4.65                                                                              10.sup.3.84                                                                        10.sup.2.26                               ______________________________________                                    

These tests thus confirmed the virucidal activity of benzalkoniumfluoride at the following concentrations (m/V) and contact times: 0.01%in 30 minutes on Polio 1 enterovirus; 0.02% in 120 minutes on Type 5adenovirus; 0.01% in 5 minutes on Herpes Simplex Virus Type 1; 0.01% in15 minutes on Herpes Simplex Virus Type 2 at 32° C. following StandardAFNOR T72-180.

Example 2

The virucidal activity of benzalkonium fluoride was tested on the HIV-1virus (LAI strain) responsible for AIDS by the Institut Pasteur usingthe infectiousness test on primary culture of activated humanlymphocytes test.

The study was carried out in two steps: we first treated the virus witha solution of benzalkonium fluoride (BKF) at 20 μg/ml, then we studiedthe residual infectiousness of the treated virus on a primary culture ofactivated human lymphocytes.

For treatment of the virus, the contact time at room temperature was 30minutes and 60 minutes, 0.9 ml of the virus added to 100 μl of BKF wasleft for 30 minutes at room temperature, then diluted in 10 ml of NTEbuffer. The sample obtained is referenced REV7. Sample REV8 correspondsto 0.9 ml of the virus added to 100 μl of BKF left for 60 minutes atroom temperature then diluted in 10 ml of NTE buffer. After dilution inthe buffer, the samples were centrifuged for 35 minutes at 40,000rev/min in order to eliminate the product possibly toxic for theinfectiousness test cells, and to concentrate the residual infectiousvirus. After centrifuging, the residue was resuspended in 1 ml ofculture medium, sealed and stored at -80° C.

Samples REV1, REV2 and REV3.1 were the positive controls for theexperiment, REV1 corresponding to 0.9 ml of viral suspension frozen at-80° C., REV2 corresponding to 0.9 ml of viral suspension diluted andcentrifuged in the same way as samples REV7 and REV8 in order toevaluate the loss of infectiousness during the centrifuging stage.REV3.1 corresponded to 0.9 ml of viral suspension left at roomtemperature for 60 minutes (maximum duration of the study) then dilutedin NTE buffer and centrifuged in the same way as the treated samples, inorder to evaluate the loss of infectiousness due to the experimentalconditions used for BKF.

A sample, REV4, of 1 ml of BKF at 20 μg/ml diluted in 10 ml of NTEbuffer and centrifuged under the conditions described for the treatedvirus, was used to control any possible toxicity on the infectiousnesstest cells due to traces of residual product not eliminated duringcentrifuging. This sample was thus used as negative control with thenon-infected infectiousness test cells.

The tests in the real sense consisted in searching for the residualinfectious power of the treated virus on activated human PBMClymphocytes. To do so, the lymphocytes, which were previouslystimulated, were incubated with 1 ml of each virus sample, treated ornot, diluted by 10 in 10 (from 10⁻⁴ to 10⁻⁷ for the negative controlsand from 10⁻¹ to 10⁻⁵ for the samples of treated virus). The cells werecentrifuged and resuspended at 10⁶ cells per ml in a complete medium.Every three or four days, the cells were counted, reseeded and thesupernatants stored at -80° C. After analysis of the results, a secondexperiment was carried out in an identical way, inoculating the cellswith half dilutions of the samples of virus treated with BKF.

Next, we measured the activity of reverse transcriptase (RT) in thesupernatants of the cell cultures. The supernatants saved on eachcellular passage (days n° 3, 7, 10, 14, 17, 21, 24 for the firstexperiment and 3, 6, 9, 13, 16, and 20 for the second experiment) weretested and their reverse transcriptase activity was quantified afterconcentration by ultracentrifuging.

We also quantified the HIV-1 antigen P25 using the Elisa "Sandwich"technique on the same supernatants culture on days n° 3, 10, 17 and 24for the first experiment and 3, 9, 16 and 20 for the second experimentafter inoculation of the cells. The ELAVIA Ag1 kit available from theSociete Diagnostics Pasteur was used. We noted that control samples REV4were not toxic for the infectiousness test cells.

Table 6 gives the results obtained for reverse transcriptase activitywith the control samples.

                                      TABLE 6                                     __________________________________________________________________________    VIRUCIDE ACTIVITY TEST OF KBF                                                 ON PRIMARY CULTURE OF ACTIVATED HUMAN LYMPHOCYTES                             TITRATION IN DILUTION OF CONTROL VIRAL SAMPLES                                         RT activity at different days (cpm/ml*)                              Sample                                                                             Dilution                                                                          Day 3                                                                             Day 7                                                                             Day 10                                                                            Day 14                                                                            Day 17                                                                            Day 21                                                                            Day 24                                       __________________________________________________________________________    REV1 10.sup.-4                                                                         391 4,304                                                                             26,311                                                                            47,192                                                                            5,529                                                                               888                                                                               712                                        control                                                                            10.sup.-5                                                                         1,354                                                                             1,786                                                                             5,417                                                                             36,738                                                                            8,450                                                                             7,157                                                                             3,854                                        positive                                                                           10.sup.-6                                                                         335 652 2,459                                                                             38,850                                                                            23,062                                                                            5,611                                                                             3,368                                             10.sup.-7                                                                         490 1,121                                                                             6,521                                                                             5,940                                                                             2,313                                                                             1,014                                                                             1,370                                        REV2 10.sup.-4                                                                         285 590 1,345                                                                             9,772                                                                             4,075                                                                             1,888                                                                             2,083                                        ctrl cen-                                                                          10.sup.-5                                                                         713 294   296                                                                               555                                                                             9,348                                                                             12,302                                                                            5,043                                        trifuging                                                                          10.sup.-6                                                                         308 351   414                                                                               326                                                                             3,509                                                                             1,568                                                                               653                                        REV3.1                                                                             10.sup.-4                                                                         203 582   569                                                                             8,425                                                                             3,703                                                                             3,778                                                                             4,559                                        exp.sup.al ctrl                                                                    10.sup.-5                                                                         572 505   577                                                                             1,395                                                                             3,155                                                                             12,243                                                                            4,607                                        60 min.                                                                            10.sup.-6                                                                         440 339 1,165                                                                             1,250                                                                               714                                                                               653                                                                               994                                        non-infected cells                                                                     247 258   363                                                                               291                                                                               630                                                                               630                                                                               366                                        __________________________________________________________________________     *cpm/ml: reverse transcriptase activity (RT) in 1 ml of culture               supernatant. The viral production is considered to be positive for a RT       activity of more than 5.000 cpm/ml (bolt type numbers)                   

We note in Table 6 that 14 days after infection, the cells inoculatedwith dilutions less than or equal to 10-7 of sample REV1 or with thedilution of 10-4 of samples REV2 and REV3.1 have viral particles in theculture supernantants. Under experimental conditions, the strength ofthe virus is defined as the inverse of the last dilution being 100%infectious. The strengths of the control samples are, respectively, 107UI/ml for REV1 and 104 for REV2 and REV3.1. A reduction of infectiousstrength is thus noted for the experimental controls. It is of alogarithmic factor of the order of 3 for the centrifuged sample, and forthe control virus left for one hour at room temperature and centrifuged.In FIG. 1, we note that no reverse transcriptase activity is revealed inthe culture supernantants inoculated with a 1/10 dilution of virussamples treated with BKF for 30 or 60 minutes. These results are to becompared to those obtained with control REV3.1 which represents aninfectious strength of 104 UI/ml.

Taking into account these results, a second experiment was carried outby inoculating PBMC lymphocytes with 1/2 dilution of viral samplestreated with BKF in order to confirm the absence of infectious virusesin the samples. The results of RT activity titration in the culturesupernatants are represented in FIG. 2. No RT activity was detected,while control virus sample REV3.1 showed positive at dilution 10-5.

FIG. 3 represents the results of titration of antigen P25 in the controlviral samples. The amount of antigen P25 is expressed in pg/ml and isevaluated using standard curve established from a reference antigensolution supplied by the manufacturer. The strengths evaluated 16 daysafter infection were 10⁷ UI/ml for REV1, 10⁵ UI/ml for REV2 and 10⁴UI/ml for REV3.1.

FIG. 4 gives the strength in P25 antigens on the lymphocyte supernatantsinoculated with 1/2 dilutions of viral samples treated with BKF. We notethat samples REV7 and REV8 are negative for antigen P25. These resultsthus confirm those obtained by following the viral expression by reversetranscriptase activity strength in the supernatants of the culturesexposed to treated or untreated virus.

Thus, these trials show that benzalkonium fluoride at 20 μg/ml induces aloss of infectious capacity in the HIV-1 virus in vitro by a logarithmicfactor of 5 for activated human lymphocytes. Thus benzalkonium fluorideat 20 μg/ml proves to be a virucide on the AIDS virus in vitro.

Example 3

Local and general tolerance of administration of benzalkonium fluorideintravenously was tested on dogs.

Two beagles, one male and one female, of an initial weight of around 10kg, 6 months old, in good health, wormed and vaccinated, were placed inan animal house. They were submitted to an intravenous treatment of 2 mgper kg per day of benzalkonium fluoride diluted at 2.10 g/l in anisotonic solute of sterile apyrogenic sodium chloride. The solution waspackaged in 10 ml ampoules and 0.95 ml per kg per day was injectedrotating the injections on the vein of each leg. The injection wascarried out using an epicranian micro-perfuser mounted on a plasticsyringe, and the administration was slow intravenous over around 90seconds. The treatment was given for 28 days. Urine tests were performedbefore the start of treatment, at the middle (after 14 days) and at theend of the treatment.

Also, blood samples were taken from the jugular before startingtreatment, after 14 days and after 28 days of treatment. H.aebutted.matological examination and complete biochemical titration werecarried out on these samples.

At the end of the treatment, the animals were killed, and we proceededwith anatompathological examinations and weighing of the followingorgans: liver, kidneys, spleen, adrenal, testicles, ovaries, thymus,thyroids. We also proceeded with histological examinations on most ofthe vital organs.

No modification of sympomatological behaviour was observed following thetreatment. During the first two weeks, the injections could be givenwithout difficulty. After, an oedema appeared.

The body weight of the animals remained stable during the entireexperimental period. The rectal temperature of the male remained stableand that of the female dog increased 1° C. during the experiment. Nonotable variation in blood pressure or heart rate was observed. Also,the various electrocardiographic parameters remained stable throughoutthe entire experimental period.

For the female dog, urine samples showed no signs of h.ae butted.molysisat the start, in the middle and at the end of treatment. For the maledog, we noted a high level of blood in the urine at the start oftreatment. The dog was left in a diuresis cage for 48 hours, after whichthe urine showed an absence of blood and traces of ketonic bodies,absence of glucose and traces of proteins. The quantities of sodium andpotassium showed variations for the male dog but remained almost stablefor the female. At the end of treatment, no trace of blood was found inthe urine taken from the bladder of the male dog at autopsy time.

Among the h.ae butted.matological parameters, only the number ofleukocytes increased after two weeks for the female dog and after fourweeks for the male. This increase corresponded to the appearance ofoedemas in a leg.

In the biochemical parameters, an increase in alkaline phosphatase andLDH was noted. No other significant variation was noted.

Histological examination of the various organs showed no alteration dueto treatment other than local reactions at the injection sites. Also,despite the severity of the treatment, no remarkable toxic effect ofbenzalkonium fluoride injected intravenously showed up.

Example 4

The high toxicity of benzalkonium fluoride in unique administration inmice and guinea pigs was determined by the Behrens and Karber method, byintracardiac, intravenous and intraperitoneal injection. The animalsused were young adult albino mice and albino guinea pigs. Ten animalswere used for each dose administered, five males and five females. Thebenzalkonium fluoride was used in a 3 g/l isotonic solution of sterileapyrogenic sodium chloride. We observed the animals for 7 days afterunique administration carried out using a sterile needle adapted to asyringe graduated in 1/10 ml to allow the treatment of each animal withan exact dose. The animals were weighed each day during the observationperiod.

The LD 50 (lethal dose 50) was determined. The results are given inTable

                  TABLE 7                                                         ______________________________________                                                               intraperi-                                             LD 50 (mg/ml)                                                                             intravenous                                                                              toneal   intracardiac                                  ______________________________________                                        Male mouse  33.24      33.10    --                                            Female mouse                                                                              36.60      34.60    --                                            Male guinea --         28.50    15.50                                         pig                                                                           Female guinea                                                                             --         20.50    16.50                                         pig                                                                           ______________________________________                                    

Thus we note that benzalkonium fluoride by intravenous, intraperitonealor intracardiac injection has a sufficiently low toxicity for us toenvisage its administration by intravascular injection at doses where itis nevertheless active, particularly at 20 to 23 mg/l of blood of thepatient to be treated.

Example 5

In vivo trials were carried out on dogs suffering from parvovirus. Thesedogs were treated with slow perfusion of benzalkonium fluoride C14dissolved at 2.1 g/l in an isotonic solute of sterile apyrogenic sodiumchloride.

A male German Shepherd weighing 20 kg, eight months old, suffering fromparvovirus (gastro-enteritis with dehydration, sero-h.ae butted.morragicenteritis with high urea level) was treated with two injections at a 12hour interval with two 10 ml ampoules. 6 days after the start oftreatment we noted both excellent tolerance and recovery of the dog.

A female German Shepherd aged 7 months and weighing 10 kg, sufferingfrom parvovirus for 4 days (gastro- enteritis with dehydration,sub-comatose state, h.ae butted.moconcentration, high urea level, h.aebutted.morragic salt) was treated with two injections separated by 12hours of the solution according to the invention. The first injectionwas 10 ml and the second 20 ml. A general associated treatment was givenfor 8 days. Tolerance to the treatment proved to be excellent and wenoted total recovery after 20 days.

A male Newfoundland of 6 months and weighing 28 kg suffering fromparvovirus was also treated with two slow intravenous injections of 10ml separated by 12 hours. At the end of the second day, the blood haddisappeared from the urine and the dog no longer vomited. After fourdays, the animal was eating normally. On the 18th day, total recoverywas noted.

A female Rottweiler aged 4 months and weighing 15 kg suffering fromparvovirus was also treated in the same way. Complete recovery was notedafter 18 days.

The same treatment was applied to a female German Pointer of 2 monthsand weighing 12 kg. The same recovery was noted (normal eating after 4days and total recovery confirmed after 18 days).

Three puppies (4 month male of 12 kg, 4 month male of 7 kg and 2 monthfemale of 6 kg) suffering from violent evolutive gastro-enteritis werealso treated with two slow intravenous injections at 12 hours interval.The puppies were suffering from sero-h.ae butted.morragicgastro-enteritis. Death occurred within two days.

Thus, apart from the three failures obtained on the very young animalssuffering from severe symptoms, the invention allowed treatment ofparvovirus in dogs.

Example 6

The invention was used to treat dogs suffering from Carre's disease.This disease, considered fatal and without treatment, is due to anultravirus.

Twelve dogs were treated with one 10 ml ampoule of benzalkonium fluoridesolution at 2.1 g/l for 20 kg of animal. The injection was given onceper day for three days. The dogs also had associated treatment(vitamins, antibiotics, vaccines, etc.).

The treatment proved effective for seven dogs who were saved and forwhom total recovery was noted after 18 days.

Among the seven dogs cured, five came from a pack in which there hadalready been two deaths, and which was greatly affected by the disease.They had already undergone an ineffective treatment. The symptoms weremainly purulent nasal mucous, cough, diarrhoea, suppuratingconjunctivitis and fever of between 40° C. and 40.5° C. average. Theefficacy of the treatment was thus particularly surprising.

The five other dogs treated all presented nervous symptoms (generalisedtrembling) and could not be treated. Consequently, the invention allowedtreatment of the cases of Carre's disease not accompanied by nervousmanifestations.

Example 7

We determined the h.ae butted.molytic concentration of benzalkoniumfluoride on total human, guinea pig and rabbit blood in vitro.

This experiment was carried out with a hospital method for looking forperipheral an.ae butted.mia. We used dilutions of a benzalkoniumfluoride solution at 3 per 1000: 300 mg/L, 150 mg/L and 75 mg/l in anisotonic solute of sterile apyrogenic sodium chloride. Each of the threedilutions was mixed at equal volume with the blood of the three species(man, guinea pig and rabbit). The mixture was slowly shaken for 5minutes thirty then centrifuged for three minutes at 3,500 rev/min. H.aebutted.molysis was shown by a more or less dark red colouring of thepersistent plasmatic supernatant. The real concentrations tested werethus 150 ppm, 75 ppm and 35.5 ppm of benzalkonium fluoride. Each testwas doubled.

Not having noted h.ae butted.molysis at 37.5 mg/l, new tests werecarried out on six samples of human blood, under the same conditions, at20 mg/L and 35 mg/l of benzalkonium fluoride.

Table 8, below, gives the results obtained.

                  TABLE 8                                                         ______________________________________                                                 Concentration of benzalkonium fluoride                                        (in ppm or mg/l)                                                              6 samples                                                                     150     75     37.5     35   20                                      ______________________________________                                        GUINEA PIG ++++      +      0      --   --                                    RABBIT     +++       +      0      --   --                                    MAN        ++        +      0      0    0                                     ______________________________________                                         + to ++++: degree of red colouring due to haemolysis                     

Thus benzalkonium fluoride at a concentration equal to or less than 20mg/l does not provoke h.ae butted.molysis in human blood, and this witha wide margin of security.

According to the invention, we may nevertheless easily check the h.aebutted.molytic concentration of benzalkonium fluoride on the blood ofthe patient to be treated before injection.

Example 8

We treated patients suffering from AIDS in the evolutive or terminalphase with a solution consisting of 2.1 g/l of benzalkonium fluoride.Before injection, the h.ae butted.molytic concentration of benzalkoniumfluoride was determined and we verified that it was well above that ofthe solution used.

A male of 37 years, weighing 70 kg, presented the following symptoms:asthenia, weight loss, bucal candidiasis, fever with a plateau of 39.7°C., ELISA (enzyme linked immunosorbant assay) test and WESTERN BLOT(confirmation test) positive, number of CD₄ (receptors at the surface ofT lymphocytes responsible for regulation of the immune response) lessthan 200 (the normal value being of the order of 1,000). The patient wastreated with slow intravenous injections of 50 ml of benzalkoniumfluoride C₁₄ at 2.1 g/l repeated every twelve hours in cures of threedays separated by five days of observation. Despite pain at theinjection point which appeared at the end of the second cure,observation at one month and seven months after treatment showed a goodgeneral state of the patient who could take up his professionalactivity. The number of CD₄ had risen to 600.

A male of 28 years, weighing 57 kg, presented the following symptoms:asthenia, weight loss, vomiting, diarrhoea, bucal candidiasis, feverwith a plateau of 39.5° C., totally beddriden patient ELISA and WESTERNBLOT tests positive, number of CD₄ equal to 20. This patient underwenttwo cures of three days as described above. On the tenth day after thestart of treatment, we noted a clear improvement in the general state,disappearance of the fever, vomiting and diarrhoea and weight gain (59.6kg). The patient could get up and walk.

A male of 44 years, weighing 78 kg, presented the following symptoms:asthenia, anorexia, insomnia, fever with a plateau of 39.7° C., ELISAand WESTERN BLOT tests positive, number of CD₄ at 650. The patient wastreated with slow intravenous injections of 50 ml of benzalkoniumfluoride C₁₄ at 2.1 g/l repeated every eight hours for three days with acomplementary antibiotic treatment (based on Doxycycline) of twocapsules each evening.

On the sixth day after the start of treatment, the fever haddisappeared. Two months after the start of treatment, we noted aprogressive disappearance of the asthenia. At the fourth month, thepatient had returned to a normal general state and taken up his militaryactivity.

A male of 42 years, weighing 52 kg, presented the following symptoms:asthenia, anorexia, sweating, inguinal adenopathy, diarrhoea,cephalagia, bucal candidiasis, fever with a plateau of 39° C., ELISA andWESTERN BLOT tests positive, number of CD₄ at 200. The patient wastreated with a cure of three days of slow intravenous injections ofbenzalkonium fluoride C₁₄ at 2.1 g/l every twelve hours. On the thirdday, a clear fall in the fever was noted. Three and a half months afterthe start of treatment, the patient presented a good general state, aweight of 56 kg, the number of CD₄ had risen to 700 and there were noclinical symptoms.

Example 9

We determined the activity in vitro of benzalkonium fluoride on themycobacterium tuberculosis (wild strain).

This experiment was carried out with the BOWEINSTEIN JENSEN mediumsurface impregnation method. The quantity of active substance absorbedby the medium is expressed in cm².

The volume of the appropriate dilution of benzalkonium fluoride was 0.25ml per tube of medium in order to arrive at concentrations ofbenzalkonium fluoride of 0.1, 0.2 and 0.3% per cm² surface ofBOWEINSTEIN JENSEN medium.

A control tube, without benzalkonium fluoride, was used for each of theseries.

Three series of tests were carried out with dilutions of 0.1, 0.01 and0.001 of the mother solution of the Bacterium Tuberculosis strainprepared at 1 mg/ml.

All the tests were carried out in double.

After four weeks of incubation at 37° C., the results showed aninhibition of the culture with concentrations of benzalkonium fluorideof 0.1, 0.2 and 0.3% per cm² surface of BOWEINSTEIN JENSEN medium, andthis was observed for the three dilutions at 0.1, 0.01 and 0.001 of theBacterium Tuberculosis mother culture.

Table 9, below, gives the results obtained.

                  TABLE 9                                                         ______________________________________                                               BK   BK       BK     BK     BK   BK                                           0.1  0.1      0.01   0.01   0.001                                                                              0.001                                 ______________________________________                                        CONTROL  (+++)  (+++)    (+++)                                                                              (+++)  (+++)                                                                              (+++)                               BKF      (-)    (-)      (-)  (-)    (-)  (-)                                 0.1%/cm.sup.2                                                                 BKF      (-)    (-)      (-)  (-)    (-)  (-)                                 0.2%/cm.sup.2                                                                 BKF      (-)    (-)      (-)  (-)    (-)  (-)                                 0.3%/cm.sup.2                                                                 ______________________________________                                         (+++) = positive culture                                                      (-) = negative culture                                                   

We claim:
 1. Solution administrable by intravascular injection in ahuman or an animal which comprises an effective amount up to 0.23% byweight of a benzalkonium fluoride having the formula: ##STR3## where Ris an alkyl radical which may vary between C₈ H₁₇ and C₁₈ H₃₇, dissolvedin an excipient injectable intravascularly.
 2. Solution according toclaim 1, which comprises about 0.2% by weight of benzalkonium fluoride.3. Solution according to claim 1, which contains at least about 0.05% byweight of benzalkonium fluoride.
 4. Solution according to claim 1,wherein R is between C₁₂ H₂₅ and C₁₄ H₂₉.
 5. Solution according to claim1, wherein R is C₁₄ H₂₉.
 6. Solution according to claim 1, packaged in 5to 15 ml ampoules, containing between 0.5 and 23 mg of benzalkoniumfluoride.
 7. Solution according to claim 1, packaged in 10 ml ampoules,containing about 21 mg of benzalkonium fluoride.
 8. Solution accordingto claim 1, which consists of benzalkonium fluoride dissolved in aphysiological solvent.
 9. Solution according to claim 1, which furthercontains at least one metallic derivative of fluorine.
 10. Solutionaccording to claim 9, wherein said derivative is lithium fluoride. 11.Method of treatment of viral or infectious diseases in a human or ananimal, which comprises administering by intravascular injection asolution of at least one benzalkonium fluoride of the formula: ##STR4##where R is an alkyl radical which may vary between C₈ H₁₇ and C₁₈ H₃₇,dissolved at an effective concentration of up to 0.23% by weight in anexcipient injectable intravascularly, wherein the disease is induced bypicornavirus, adenovirus. herpetovirus, HIV virus, parvovirus or isCarre's disease.
 12. Method according to claim 11, wherein said solutionis an intravascular injection solution comprising about 0.2% by weightof benzalkonium fluoride.
 13. Method according to claim 11, wherein theintravascular injection solution comprises more than 0.005% by weight ofbenzalkonium fluoride.
 14. Method according to claim 11, wherein theconcentration of benzalkonium fluoride administered by intravascularinjection is delivered by slow injection of 0.5 to 23 mg of benzalkoniumfluoride per liter of blood of the patient treated.
 15. Method accordingto claim 11, wherein the concentration of benzalkonium fluorideadministered by intravascular injection is delivered by slow injectionof 21 mg of benzalkonium fluoride per liter of blood of the patienttreated.
 16. Method according to claim 11, wherein R is between C₁₂ H₂₅and C₁₄ H₂₉.
 17. Method according to claim 11, wherein the intravascularinjectable solution comprises between 0.5 to 23 mg of benzalkoniumfluoride dissolved in between 5 and 15 ml of solvent.
 18. Methodaccording to claim 11, wherein the intravascular injectable solutioncomprises 21 mg of benzalkonium fluoride dissolved in 10 ml of solvent.19. Method according to claim 11, wherein the intravascular injectablesolution comprises benzalkonium fluoride dissolved in a physiologicalsolvent.
 20. Method according to claim 11, wherein the intravascularinjectable solution comprises at least one metallic fluorine derivative.21. Method according to claim 20, wherein said derivative is lithiumfluoride.
 22. Method of treatment of AIDS in the declared or evolutivephase, which comprises administering to a human suffering from AIDS inthe declared or evolutive phase by intravascular injection a solution ofat least one benzalkonium fluoride of the formula: ##STR5## where R isan alkyl radical which may vary between C₈ H₁₇ and C₁₈ H₃₇, dissolved atan effective concentration of up to 0.23% by weight in an excipientinjectable intravascularly.
 23. Method of treatment of tuberculosis,which comprises administering to a human suffering from tuberculosis byintravascular injection a solution of at least one benzalkonium fluorideof the formula: ##STR6## where R is an alkyl radical which may varybetween C₈ H₁₇ and C₁₈ H₃₇, dissolved at an effective concentration ofup to 0.23% by weight in an excipient injectable intravascularly.